The emergence of multiple drug resistant pathogens is becoming problematic worldwide, and the discovery of new antibiotics continues to decline. The broad, long-term objective of this proposal is to discover, characterize, and develop a new structural class of antibiotics-nucleoside antibiotics-that target bacterial translocase I involved in peptidoglycan cell wall biosynthesis. The specific aims of this proposal are (I) to characterize the assembly and incorporation of the aminoribosyl moiety found within the family of lipopeptidyl-nucleoside antibiotics and (II) to functionally and mechanistically characterize enzymes catalyzing novel or unusual biochemical reactions represented herein by a new family of serine hydroxymethyltransferase-like enzymes that are hypothesized to catalyze an aldol-type condensation to form unusual nonproteinogenic amino acids. Specific Aim I and II will be achieved by using the robust genetic system developed for the lipopeptidyl nucleoside A-90289-producing strain for in vivo studies employing gene inactivation and cross-complementation, and recombinant proteins will be exploited for functional and mechanistic studies in vitro. The results will establish a new mechanism for incorporating ribosyl units into natural product scaffolds and will establish a paradigm for the entry into high-carbon nucleoside antibiotics. The results will be essential for our long-term goals of searching for new nucleoside antibiotics using genetic information (a genotype-to-chemotype approach), will allow for the structural diversification of the parent scaffolds using combinatorial biosynthesis and total synthesis, and will provide the basis for the design of second generation antibiotics with improved biocompatibility and pharmacological properties. PUBLIC HEALTH RELEVANCE: Diseases caused by multidrug resistant bacteria are becoming a significant threat to human health worldwide. The goal of this proposal is to study nucleoside antibiotics that represent a new structural class of antibiotics, have a different mode of action than clinically used antibacterial drugs, and are in general not toxic to animals.